Benzidine has been used in a method for the microdetermination of hemoglobin in blood plasma, serum, or other fluids, as in the procedure of Crosby and Furth described in Blood 11:380 (1956). However, benzidine is extremely carcinogenic, and thus has been recently rendered unavailable for routine laboratory use.
As reported by Holland, et. al. in Tetrahedron 30:3299 (1974) it has been proposed to substitute for benzidine the compound 3,3'5,5'-tetramethylbenzidine, which appears to be non-carcinogenic, and yet which, in the presence of hydrogen peroxide, forms a highly-colored complex with hemoglobin. However, the procedure, as suggested by Holland, et al. is insufficiently sensitive to quantitatively determine trace amounts of hemoglobin on the order of 10 milligram percent and less.
Accordingly, the Holland, et al. test is generally unsuitable for testing for the presence of trace amounts of hemoglobin in blood plasma taken from stored blood, for example as a determination of the viability of the bood. As blood deteriorates, hemoglobin is released into the plasma by dying blood cells. Thus a quantitative analysis of the hemoglobin present in such plasma is an index of the viability of the blood.
In accordance with this invention, a significant improvement in sensitivity has been accomplished by performing the hemoglobin test with a benzidine derivative and hydrogen peroxide in a mixture at an acid content which is greater than that which has been used in the prior art relating to hemoglobin testing with this benzidine derivative. The result of this appears to be the production of a different, green complex material with hemoglobin (the hemoglobin complex in the prior art is blue or blue-green) which is capable of being analyzed in concentrations as low as on the order of 1 milligram of hemoglobin per 100 ml. of the solution to be tested. This is indeed far lower than that available for the prior art. Furthermore, this invention can be utilized to provide a complex which has good stability at the endpoint, to permit reading of the endpoint over a significant period of time, rather than one specific moment.
By this invention, the sensitivity of the method is adjustable so that the quantitative presence of hemoglobin can be determined photometrically over a significant, highly dilute range of concentrations, for example, from 1 to 30 mg. of hemoglobin per 100 ml. of solution, by proper adjustment of the pH and other factors.